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2019

Hematology, plasma biochemistry, and hormonal analysis of captive pine ( ruthveni): effects of intrinsic factors and analytical methodology

Luca Giori University of Tennessee, Knoxville

Nicole I. Stacy University of Florida, Gainesville

Michael Ogle Zoo Knoxville, 3500 Knoxville Zoo Drive, Knoxville, TN 37914, USA

Stephen Nelson Zoo Knoxville, 3500 Knoxville Zoo Drive, Knoxville, TN 37914, USA

Kellie A. Fecteau University of Tennessee, Knoxville

See next page for additional authors Follow this and additional works at: https://trace.tennessee.edu/utk_compmedpubs

Recommended Citation Giori, Luca; Stacy, Nicole I.; Ogle, Michael; Nelson, Stephen; Fecteau, Kellie A.; and Cushing, Andrew C., "Hematology, plasma biochemistry, and hormonal analysis of captive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic factors and analytical methodology" (2019). Faculty Publications and Other Works -- Biomedical and Diagnostic Sciences. https://trace.tennessee.edu/utk_compmedpubs/129

This Article is brought to you for free and open access by the Veterinary Medicine -- Faculty Publications and Other Works at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Faculty Publications and Other Works -- Biomedical and Diagnostic Sciences by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Authors Luca Giori, Nicole I. Stacy, Michael Ogle, Stephen Nelson, Kellie A. Fecteau, and Andrew C. Cushing

This article is available at TRACE: Tennessee Research and Creative Exchange: https://trace.tennessee.edu/ utk_compmedpubs/129 Hematology, plasma biochemistry, and hormonal analysis of captive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic factors and analytical methodology Luca Giori, Nicole I. Stacy, Michael Ogle, Stephen Nelson, Kellie A. Fecteau & Andrew C. Cushing

Comparative Clinical Pathology

ISSN 1618-5641

Comp Clin Pathol DOI 10.1007/s00580-019-03030-w

1 23 Your article is published under the Creative Commons Attribution license which allows users to read, copy, distribute and make derivative works, as long as the author of the original work is cited. You may self- archive this article on your own website, an institutional repository or funder’s repository and make it publicly available immediately.

1 23 Comparative Clinical Pathology https://doi.org/10.1007/s00580-019-03030-w

ORIGINAL ARTICLE

Hematology, plasma biochemistry, and hormonal analysis of captive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic factors and analytical methodology

Luca Giori1 & Nicole I. Stacy2 & Michael Ogle3 & Stephen Nelson3 & Kellie A. Fecteau1 & Andrew C. Cushing4

Received: 7 March 2019 /Accepted: 17 July 2019 # The Author(s) 2019

Abstract Blood analyte data are useful in health assessments and management of . There is a knowledge gap for blood analyte data of the endangered Louisiana pine (LPS; Pituophis ruthveni). The objectives of this study were to provide baseline hematology, plasma biochemical, and hormone data of captive LPS, to compare the data in juvenile and adult snakes and in adult snakes by sex, and to investigate methodological differences in hormone (serum vs. plasma) and protein analyses (total solids versus total protein). Blood samples from apparently healthy captive LPS were analyzed for hematology and plasma biochemistry (n = 11) and plasma and serum hormone analyses (n = 9). Packed cell volume (PCV) and absolute heterophils were significantly higher in adult compared with juvenile LPS, while PCV, white blood cell count, and absolute lymphocytes were higher in adult males compared with adult females. Significantly higher plasma concentrations were found in adults compared with juveniles for calcium, total protein, total solids, albumin, globulins, and bile acids. No significant differences were observed in 17β-estradiol measured in serum and plasma when comparing adults and juveniles and for 17β-estradiol in adult males and females. Plasma concentrations of 17β-estradiol were significantly lower than in serum. Serum testosterone in two adult males was 8.33 and 35.53 nmol/L, respectively, while it was undetectable in females and juveniles (n = 5). This study is the first to provide baseline information on blood analytes in endangered LPS, which will be useful for individual in managed care and as baseline for future population-level assessments.

Keywords Blood . Methodology . Plasma chemistry . Hormones .

Introduction

Pituophis is a genus of non-venomous large-bodied constric- Electronic supplementary material The online version of this article tor snakes belonging to the family, which is con- (https://doi.org/10.1007/s00580-019-03030-w) contains supplementary sidered the largest and most structurally diverse snake group, material, which is available to authorized users. including about 50% of all known living snake (Uetz et al. 2018; Vitt and Caldwell 2014). Previously endemic to * Luca Giori [email protected] western Louisiana and eastern Texas, the Louisiana pine snake (LPS; Pituophis ruthveni) population has declined precipi- tously in recent decades and is now restricted to highly 1 Department of Biomedical and Diagnostic Sciences, College of fragmented and isolated patches of both southeastern states Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN 37996, USA after degradation of its natural habitat, mostly due to intensive silviculture, and is thus categorized as endangered by the 2 Department of Comparative, Diagnostic, and Population Medicine, College of Veterinary Medicine, University of Florida, 2015 SW 16th IUCN Red List (International Union for Conservation of Ave, Gainesville, FL 32608, USA Nature 2019; Rudolph et al. 2006). 3 Zoo Knoxville, 3500 Knoxville Zoo Drive, Knoxville, TN 37914, Hematological and biochemical analytes are essential base- USA line diagnostics in the health assessment of any species. There 4 Department of Small Clinical Sciences, University of is extensive variation in such data between species and even Tennessee, 2407 River Drive, Knoxville, TN 37996, USA genus, and published reference data are lacking for many Comp Clin Pathol snake species. In addition, significant variation can occur from their tubs to the zoo clinic in individual cotton bags, were within a given species due to intrinsic and extrinsic factors weighed, and a physical examination was performed by one of such as ambient environment, season, analytical methodolo- the authors (AC). All snakes were physically examined and gy, physiological state, and diet (Campbell 2015c; Stacy et al. found to be apparently healthy (e.g., normal feeding behavior, 2011). Hormone analysis in snakes is not routinely performed absence of external injuries or lesions) and were not on any due to lack of readily validated assays for diagnostic use and medications for at least 4 weeks prior to sampling. Total length thus most frequently used in research studies offering insights (TL) was also recorded. The sex of each snake had previously into reproductive physiology, breeding suitability, or timing been assessed by probing. and may offer an alternative tool for sexing as demonstrated in previous studies (Edwards and Jones 2001; Jones 2011; Blood collection Taylor and DeNardo 2011; Taylor et al. 2004). Published literature about LPS is primarily focused on bi- Blood was collected by venipuncture of the ventral coccygeal ological investigations on natural habitat, growth rates, and vein using a 22-gauge needle attached to a 3-mL syringe. In ecological behaviors of the species (Ealy et al. 2004;Himes order to facilitate blood collection from the tail, the snakes 2001; Himes et al. 2002;Pierceetal.2014; Rudolph et al. were restrained with the head elevated above the level of the 2007). To the authors’ knowledge, information on hematolo- tail. For two smaller snakes, a cardiac puncture was elected in gy, plasma biochemistry, and hormone data in this species is order to obtain the minimum amount of blood to perform the unavailable to date. Blood analyte data can provide useful analysis. Immediately after collection, blood was aliquoted information for answering biological and ecological questions into three different tubes: one heparinized tube without gel regarding an individual and even a population if baseline data separator (BD Microtainer® lithium heparin tube, Franklin with comparable methodology is available for reference. The Lakes, NJ, USA) used for hematological evaluation, one hep- objectives of this study were to (1) provide baseline hemato- arinized tube with gel separator (BD Microtainer® PST™ logical, plasma biochemical, and hormone data of captive lithium heparin tube, Franklin Lakes, NJ, USA) used for plas- LPS, to (2) compare the data in juvenile and adult snakes ma biochemistry, and one spray-coated silica tube for serum and in adult snakes by sex, and to (3) investigate methodolog- (BD Vacutainer® Plus plastic serum tube, Franklin Lakes, NJ, ical differences in hormone (serum vs. plasma) and protein USA) used for hormone analysis. Two blood films were im- analyses (total solids versus total protein). mediately prepared by author LG from each heparinized blood sample and allowed to air dry.

Materials and methods Hematological and plasma biochemistry analysis

Eleven captive LPS housed at Zoo Knoxville, TN, USA, were Blood samples were processed and analyzed at the University included in this study. The apparently healthy snakes included of Tennessee College of Veterinary Medicine Clinical five juveniles (< 18 months) and six adults (8–12 years). Pathology Laboratory (UTCVM-CPL) within 3 h of collec- Juvenile and adult weights ranged from 275 to 440 g and from tion for routine hematology and plasma biochemical analysis. 1490 to 4200 g, respectively. Snout-vent length (SVL) and In addition, an equal volume of 25 μL of whole blood and total length (TL) varied from 87 to 101.5 cm and from 97 to supravital dye New Methylene Blue (NMB) were mixed and 114 cm for the juveniles and from 136 to 198 cm and from 158 incubated for 15 min for reticulocyte counts (No. to 230 cm for adults. reticulocytes/1000 red blood cell (RBC)) that were performed The snakes were housed in a rack system and continuously by one author (LG) following the standard protocol used at the maintained at 25–30 °C. The adults were provided with a UTCVM-CPL. For routine hematological analysis, heparin- 34 °C basking spot from 1st March to 15th December each ized blood was spun at 16,128 g for 10 min (PowerSpin MH year. From mid-December to the end of February, adults are in Centrifuge, Unico, Dayton, NJ) in capillary tubes to obtain the brumation and the temperature was maintained between 10 spun packed cell volume (PCV). A white blood cell (WBC) and 13 °C to mirror natural habitat conditions. Juveniles were count was performed by an experienced technologist using the fed weekly with an appropriately sized . The adults semidirect technique using phloxine B (Avian Leukopet, were fed two to three small or medium rats in the spring for Vetlab Supply, Palmetto Bay, FL, USA) as previously de- the upcoming breeding season and then one to two small or scribed (Campbell 2015b; Geffre et al. 2009b; Rush et al. medium rats weekly throughout the rest of the active season. 2016). Both blood films were stained with Wright–Giemsa For all snakes, the length of the artificial daylight is 9 h year- stain with an automatic methanolic slide stainer (Mod.7151, round. The snakes were sampled as part of routine examina- Wescor, Inc., Logan, UT). The same technologist performed tions on the same day in fall, which is outside the known WBC differential, WBC morphology, RBC morphology, breeding season for this species. Snakes were transported thrombocyte estimate, and hemoparasite review on blood Comp Clin Pathol smears. Leukocytes were classified as heterophils, lympho- 104%, while recovery of a known amount of 17β-estradiol cytes, monocytes, eosinophils, basophils, and azurophils. assay standard added to snake serum was 92%. Thrombocytes were assessed as “adequate” or “not adequate” The limit of detection for 17β-estradiol, progesterone, and based on the size and number of thrombocyte clumps. Blood testosterone were < 36.71 pmol/L, < 0.64 nmol/L, and < cell sizes were evaluated using an eyepiece graticule with a 0.12 nmol/L, respectively. scale previously calibrated by a stage micrometer. After centrifugation of the heparinized blood at 700×g rpm for 10 min (Sero-Fuge centrifuge, Becton, Dickinson and Co, Statistical analysis Franklin Lakes, NJ), plasma was separated and transferred in specific microcups for biochemistry analysis with a Cobas Biochemistry, hematology, and hormone data were analyzed c501 analyzer (Roche Diagnostics, Indianapolis IN, USA). using the non-parametric Kruskal-Wallis test to detect the ef- Total solids (TS) were assessed using a refractometer fects of gender in adults and age (i.e., adult vs. juvenile). The (Mod.1310400A, Reichert Analytical Instruments, Depew, presence of a single male in the juvenile group precluded NY). The UTCVM-CPL biochemistry panel for reptiles in- analysis by sex in this age group. The difference in 17β- cluded albumin (ALB), alkaline phosphates (ALKP), aspar- estradiol concentrations between serum and plasma was ana- tate aminotransaminase (AST), bile acids (BA), total calcium lyzed using paired t test for repeated measures. Significance (CA), creatine kinase (CK), cholesterol (CHOL), globulins was identified at p< 0.05. All analyses were conducted in (GLOB), glucose (GLU), lactate dehydrogenase (LDH), SAS9.4 for Windows 64× (SAS Institute, Cary, NC). The phosphorus (P), urea (BUN), total protein (TP), and uric acid correlation between total solids by refractometer and total pro- (UA). tein concentration measured by the chemistry analyzer (biuret method) was calculated with the Spearman rank correlation Hormonal analysis method because data were not normally distributed. Correlation results were categorized as very high (r >0.90), An adequate amount of plasma in addition to serum obtained high (r = 0.70 to 0.90), moderate (r = 0.50 to 0.70), or low after centrifugation of non-heparinized blood (at 700 g for (r = 0.30 to 0.50) (Mukaka 2012). Histograms for all hematol- 12 min, Thermo Scientific IEC CL10 centrifuge, ogy and chemistry analytes are provided in the Electronic Thermofisher Scientific, Waltham, MA) was aliquoted, kept supplementary materials. Data on 17-hydroxyprogesterone at 4 °C, and transported to the UTCVM Diagnostic were excluded from the study due to all samples resulting Endocrinology Service within 30 min for analysis of the fol- below the limit of detection of the assay (0.03 nmol/L), pre- lowing hormones: 17β-estradiol, 17-hydroxyprogesterone sumptively due to lack of sufficient cross-reactivity of the (17OHP), progesterone, and testosterone. 17β-estradiol and assay. Statistical analysis was not performed on testosterone 17OHP were measured in duplicate both in serum and plasma due to female (n = 2) and juvenile (n = 3) data resulting below samples for 8 of the 11 snakes using radioimmunoassay (17β- the detection limit of the assay (< 0.12 nmol/L), but two male estradiol Radioimmunoassay, MP Biomedicals, USA; 17- samples had detectable concentrations. Due to low sample hydroxyprogesterone Radioimmunoassay, MP Biomedicals, number, we only compared estrogen concentrations from se- USA) (Tumkiratiwong et al. 2012). Progesterone and testos- rum and heparinized plasma statistically. terone were measured in duplicate both in serum and plasma samples for 8 of the 11 snakes using a chemiluminescence assay (IMMULITE® 2000XPi Immunoassay Analyzer, Results Siemens Healthcare, USA). Validation of the chemiluminescence and radioimmunoas- Results of hematological analyses are shown in Tables 1 and says was performed by determining inter- and intra-assay pre- 2. All samples had minimal trace values of lipemia, hemolysis, cision and accuracy as suggested by the American Society of and "icterus" lower than 30 (on a scale of 500 by chemistry Veterinary Clinical Pathology (ASVCP) Quality Control analyzer) except for one sample in an adult male with mild Guidelines and Lee et al. (2006). Intra- and inter-assay coef- hemolysis (114/500) which was not considered to affect ficients of variation for 17β-estradiol and progesterone assays chemistry analyte data. Additional visible evaluation did not were 4.4% and 5.8%, and 5.8% and 8.2%, respectively. show any overt plasma discolorations. Assays were validated 3 months prior to this study, using Packed cell volume was significantly higher in adults com- pooled serum obtained from fresh blood samples collected pared with juveniles (p = 0.0274) and in adult males compared from the same six LPS adults (three females and three males) with adult females (p = 0.0431). Lymphocytes were the pre- in a pilot experiment. Recovery of a known amount of pro- dominant WBC type observed in all snakes, followed by het- gesterone assay control material (Bio-Rad Lyphochek; erophils. Heterophils were significantly higher in adults com- immunoassay Plus control) added to snake serum was pared with juveniles (p=0.0441). Monocytes and azurophils Comp Clin Pathol

Table 1 Descriptive hematological and plasma biochemical data in adult (n = 6) and juvenile (n = 5) apparently healthy captive Louisiana pine snakes (Pituophis ruthveni)

Analyte Life stage Mean SD Median Min Max

PCV (L/L) Adult 0.38 ±0.5 0.385 0.29 0.43 * Juvenile 0.26 ±0.7 0.30 0.17 0.34 WBC (×109/L) Adult 13.15 ±6.01 15.75 4.90 20.7 Juvenile 6.62 ±1.33 7.20 4.50 8.30 Heterophils (×109/L) Adult 1.74 ±0.68 1.82 0.60 2.69 * Juvenile 0.74 ±0.11 0.70 0.58 0.90 Lymphocytes (×109/L) Adult 10.20 ±4.98 12.0 3.04 16.97 Juvenile 4.57 ±0.89 4.80 3.10 5.55 Monocytes (×109/L) Adult 0.50 ±0.36 0.46 0.10 1.07 Juvenile 0.46 ±0.21 0.40 0.20 0.80 Azurophils (×109/L) Adult 0.46 ±0.44 0.37 0.0 1.38 Juvenile 0.66 ±0.21 0.58 0.40 0.90 Basophils (×109/L) Adult 0.07 ±0.09 0.0 0.0 0.20 Juvenile 0.30 ±0.33 0.20 0.0 0.90 Eosinophils (×109/L) Adult 0.0 ±0.0 0.0 0.0 0.0 Juvenile 0.0 ±0.0 0.0 0.0 0.0 ALKP (nkat/L) Adult 767 ±483 600 283 1434 Juvenile 517 ±100 483 400 650 AST (nkat/L) Adult 233 ±67 200 167 350 Juvenile 83 ±350 83 33 933 LDH (nkat/L) Adult 1584 ±800 1300 733 2917 Juvenile 1300 ±1367 600 167 3934 Creatine kinase (nkat/L) Adult 7701 ±2751 6801 4701 11,919 Juvenile 10,219 ±8135 6935 1750 20,137 Phosphorus (mmol/L) Adult 1.4 ±0.3 1.3 1.1 1.7 Juvenile 1.3 ±0.3 1.1 1.0 1.8 Calcium (mmol/L) Adult 4.4 ±0.2 4.4 3.9 4.7 * Juvenile 3.7 ±0.4 3.5 3.5 4.5 Ca:P ratio Adult 3.3 ±0.6 3.3 2.5 4.1 Juvenile 3.1 ±0.4 3.2 2.5 3.7 Glucose (mmol/L) Adult 4.1 ±1.4 3.9 2.4 6.8 Juvenile 3.4 ±0.9 3.1 2.8 5.2 Total Protein (g/L) Adult 71.0 ±4.0 70.0 65.0 77.0 * Juvenile 53.0 ±5.0 51.0 48.0 63.0 Total Solids (g/L) Adult 97.0 ±7.0 98.0 84.0 105.0 * Juvenile 66.0 ±6.0 65.0 57.0 76.0 Albumin (g/L) Adult 20.0 ±1.2 20.0 18.0 22.0 * Juvenile 17.0 ±1.4 18.0 15.0 19.0 Globulin (g/L) Adult 51.0 ±3.4 50.0 47.0 58.0 * Juvenile 36.0 ±4.4 33.0 32.0 44.0 A:G ratio Adult 0.4 ±0.0 0.4 0.3 0.4 * Juvenile 0.5 ±0.1 0.5 0.4 0.6 Cholesterol (mmol/L) Adult 18.6 ±2.7 18.6 14.4 23.3 Juvenile 12.6 ±4.9 11.0 9.2 22.3 Bile Acids (mmol/L) Adult 9.7 ±4.4 10 3 15 * Juvenile 3 ±2.1 2 1 7 Uric acid (mmol/L) Adult 547.2 ±315.2 463.9 267.7 1171.8 Juvenile 267.7 ±113.0 208.2 154.6 475.8

A:G ratio, plasma albumin-to-globulin ratio; Ca:P ratio, calcium-to-phosphorus ratio; ALKP, alkaline phosphatase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase *p < 0.05, statistical significance Comp Clin Pathol were similar in all samples. No significant differences were chromatin than lymphocytes. They were often clumped in observed between adults and juveniles for WBC counts (p = blood smears due to activation during blood collection or 0.2012), lymphocytes (p=0.2012), monocytes (P=1.0000), due to heparin effects (Fig. 1i). Thrombocytes were consid- azurophils (p= 0.1190), and basophils (p= 0.1944). WBC ered adequate in blood films from all snakes. Hemoparasites counts (p = 0.0495), and lymphocytes (p = 0.0495) were sig- were absent in all samples. nificantly higher in adult males versus adult females. Results of plasma biochemical analyses are shown in The morphology of RBC was consistent with descriptions Tables 1 and 2. Significantly higher concentrations were iden- in other snakes (Campbell 2015c; Jenkins-Perez 2008; Stacy tified in adults compared with juveniles for the following et al. 2011): mature erythrocytes were ellipsoid and nucleated analytes: CA (p =0.0441);TP(p =0.0061);TS(p =0.0061); (6–8 μmwide,20–22 μm long; Fig. 1a–l) containing orange- ALB (p =0.0253);GLOB(p =0.0059);andBA(p =0.0219). pink homogeneous cytoplasm. Single small punctate baso- No statistical differences were observed for other biochemical philic inclusions most consistent with previously described analytes. The correlation between TS and TP was considered membranes of degenerate organelles were occasionally pres- high (r = 0.82). No significant differences were identified in ent (Campbell 2015c). Nuclei were centrally located, with adult males and females. irregular nuclear margins (from oval to trapezoid) and dark Results of hormone analyses are shown in Table 3. blue densely clumped chromatin. Immature erythrocytes Sufficient serum samples were obtained from four of six adult (polychromatophils by Wright-Giemsa; reticulocytes by snakes and from three juveniles. Adequate amounts of plasma NMB) were observed in adult and juvenile individuals (mean were available for four adults (for which serum was also avail- 5%, range 2–10% using NMB). Higher numbers of able) and five juveniles. When concentrations of progesterone polychromatophils, namely 8–10%, including immature pre- and testosterone were undetectable or below the limit of de- cursor stages and mitotic figures, were noticed in two juvenile tection (< 0.64 nmol/L for progesterone and < 0.12 nmol/L for individuals with lower PCVof 18 and 17%, respectively, sug- testosterone), data were excluded from the statistical analysis. gestive of an erythroid regenerative response (Fig. 1a, b, d). For this reason, due to low sample numbers, statistical analy- Since erythroid precursor stages can be misinterpreted as re- ses for comparisons of testosterone and progesterone concen- active or atypical lymphocytes, the morphology of these cells trations were not performed. No significant differences were needs to be carefully evaluated. On NMB-stained blood films, observed in serum and plasma 17β-estradiol (serum, p = residuals of RNA/cytoplasmic organelles were also observed 0.1573; plasma, p = 0.0864) between adults and juveniles. in more mature RBC as multiple punctate darker blue precip- No significant differences between adult males and females itates (Fig. 1c) scattered throughout the entire cytoplasm. A were seen in 17β-estradiol concentration in both samples (se- clear abundant reticulum of precipitate surrounding the nuclei rum, p = 0.1213; plasma, p = 0.1213). A positive correlation was apparent in earlier precursors. (r = 0.821) was found between serum and plasma sample con- Heterophils were the largest WBC (mean 21 μm; range 19– centrations for 17β-estradiol, with serum concentrations 23 μm; Fig. 1e, f) and similar in morphology as described in trending consistently significantly higher (p = 0.0156) than other snake species (Stacy et al. 2011). Heterophil left-shifting those of plasma. or evidence of toxicity were absent in blood films from all Although statistical analysis was not possible due to low sam- snakes. Lymphocytes resembled those of mammalian and oth- ple number (e.g., insufficient sample volume or values below er reptile species. Lymphocytes ranged in size from 8 to detection limit), an overt difference between testosterone concen- 14 μm(mean11μm, Fig. 1f). Larger reactive or plasmocytoid trations was observed between adult males and females. The two lymphocytes with deeply basophilic cytoplasm and pale adult males had serum (and plasma) testosterone of 35.53 (40.81) perinuclear regions were rarely observed. Monocytes (Fig. and 8.33 nmol/L (6.97), respectively, while the concentration of 1j, k) and azurophils (Fig. 1g, h) were lower in number com- serum (and plasma) testosterone for two adult females was below pared with heterophils and lymphocytes and varied in size the limit of detection of our assay (< 0.12 nmol/L). Serum tes- from mean 16 μm for monocytes and 18 μm for azurophils. tosterone concentrations were below the limit of detection in Neither melanomacrophages nor phagocytic macrophages three juvenile animals (one male, two females). Plasma testoster- were observed in any blood smears. Basophils were easily one concentrations were below detection limit in all juvenile recognizable as small round cells (mean 8 μm; range 6 to animals (four females, one male). 12 μm; Fig. 1l) packed with numerous dark blue granules obscuring the nucleus. Occasionally, ruptured basophils were seen as naked nuclei surrounded by many scattered metachro- Discussion matic granules. Eosinophils were not observed in any of the samples. Similar to other reptiles, thrombocytes in LPS had This report provides baseline hematology, chemistry, and hor- clear cytoplasm occasionally containing vacuoles and an mone data in the endangered Louisiana pine snake, and doc- ovoid/roundish nucleus with more densely clumped uments evidence that age, sex, and analytical methodology Comp Clin Pathol

Table 2 Descriptive hematological and plasma biochemical data in adult male (n = 3) and female (n = 3) apparently healthy captive Louisiana pine snakes (Pituophis ruthveni)

Analyte Sex Mean SD Median Min Max

PCV (L/L) Male 0.42 ±0.1 0.43 0.40 0.43 * Female 0.34 ±0.4 0.37 0.29 0.37 WBC (×109/L) Male 17.87 ±2.01 16.7 16.20 20.70 * Female 8.43 ±4.86 5.1 4.90 15.3 Heterophils (×109/L) Male 2.26 ±0.36 2.3 1.80 2.69 Female 1.22 ±0.51 1.23 0.60 1.84 Lymphocytes (×109/L) Male 14.26 ±1.92 13 12.80 16.97 * Female 6.15 ±3.61 4.20 3.04 11.20 Monocytes (×109/L) Male 0.57 ±0.21 0.10 0.10 1.07 Female 0.42 ±0.46 0.40 0.20 0.80 Azurophils (×109/L) Male 0.27 ±0.19 0.34 0.20 1.38 Female 0.64 ±0.53 0.58 0.40 0.90 Basophils (×109/L) Male 0.07 ±0.09 0.0 0.0 0.20 Female 0.07 ±0.09 0.0 0.0 0.20 Eosinophils (×109/L) Male 0.0 ±0.0 0.0 0.0 0.0 Female 0.0 ±0.0 0.0 0.0 0.0 ALKP (nkat/L) Male 1033 ±500 1334 333 1434 Female 483 ±267 317 283 867 AST (nkat/L) Male 233 ±83 183 167 350 Female 217 ±33 217 183 250 LDH (nkat/L) Male 1584 ±950 1100 733 2917 Female 1567 ±617 1484 867 2367 Creatine kinase (nkat/L) Male 7268 ±3300 5168 4701 11,919 Female 8135 ±1950 7335 6251 10,835 Phosphorus (mmol/L) Male 1.2 ±0.1 1.1 1.1 1.4 Female 1.5 ±0.2 1.1 1.0 1.8 Calcium (mmol/L) Male 4.5 ±0.2 4.6 4.3 4.7 Female 4.2 ±0.2 3.5 3.5 4.5 Ca:P ratio Male 3.77 ±0.35 3.93 3.28 4.08 Female 2.84 ±0.35 2.69 2.52 3.33 Glucose (mmol/L) Male 3.7 ±0.4 3.6 3.3 4.3 Female 4.5 ±1.8 4.4 2.4 6.8 Total Protein (g/L) Male 70.0 ±1.9 69.0 69.0 73.0 Female 71.0 ±4.9 70.0 65.0 77.0 Total Solids (g/L) Male 99.0 ±3.1 101.0 95.0 102.0 Female 95.0 ±8.6 95.0 84.0 105.0 Albumin (g/L) Male 20.0 ±1.2 20.0 19.0 22.0 Female 19.0 ±0.8 19.0 18.0 20.0 Globulin (g/L) Male 50.0 ±0.8 50.0 49.0 51.0 Female 52.0 ±4.6 50.0 47.0 58.0 A:G ratio Male 0.4 ±0.0 0.4 0.3 0.4 Female 0.5 ±0.1 0.5 0.4 0.6 Cholesterol (mmol/L) Male 18.3 ±0.9 18.1 17.3 19.5 Female 19.0 ±3.6 19.1 14.4 23.3 Bile acids (mmol/L) Male 11 ±3.7 12 6 15 Female 8.3 ±4.5 8 3 14 Uric acid (mmol/L) Male 374.7 ±148.7 273.6 267.7 582.9 Female 719.7 ±345.0 642.4 339.0 1171.8

A:G ratio, plasma albumin-to-globulin ratio; Ca:P ratio, calcium-to-phosphorus ratio; ALKP, alkaline phosphatase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase *p < 0.05, statistical significance Comp Clin Pathol can affect interpretation of these analytes. Hematology and gender, age, physiological status, breeding season, hiber- biochemistry data are an essential diagnostic tool for the eval- nation) and extrinsic (e.g., analytical methodology, sea- uation of the health status in any species as shown by their son, temperature, environmental conditions, husbandry, common use as components of individual or population-level diet, wild vs. captivity) factors can profoundly affect health assessment protocols. Since controlled studies are lim- blood cell proportions and influence plasma enzyme ac- ited to only some of the most commonly kept reptile species tivities and protein concentrations (Campbell 2015c). In and great variability is common even between individuals our study animals, effects of some of the extrinsic and belonging to the same genus due to many intrinsic and extrin- intrinsic factors were kept constant, since all snakes be- sic factors (Stacy et al. 2011;Uetzetal.2018), investigations long to the same zoological institution, are maintained in various reptile species will contribute to increasing the at identical environmental, nutritional, and husbandry knowledge on blood data variations. This information can conditions, and consistent analytical methodology was prove useful for various applications, such as medical treat- applied. In addition to these aforementioned factors that ment, physiology, understanding mechanisms of disease, and need to be considered in clinicopathological data inter- contributions to population-level conservation efforts. pretation, formal reference intervals should be Intrinsic and extrinsic factors are keystones as a basis established based on recommendations by the Quality for accurate interpretation of clinicopathological data Assurance and Laboratory Standards (QALS) committee notably in reptiles. The many intrinsic (e.g., species, of the ASVCP (Friedrichs et al. 2012)usingasample

Fig. 1 Image composite of blood cell types of the Louisiana pine snake (Pituophis ruthveni). a, b Erythroid precursors; c red blood cells (RBC) stained with New Methylene Blue stain showing mature RBC with punctate pre- cipitates and two immature stages with abundant reticulum staining; d mitotic figure in immature RBC and polychromatophil; e hetero- phil; f heterophil and small lym- phocyte; g, h azurophil; i throm- bocyte clump; j, k monocytes; l basophil. Wright Giemsa stain unless otherwise indicated; c, i × 50 objective; a, b, d–h, j–l ×100 objective Comp Clin Pathol

Table 3 Hormonedatafor apparently healthy, captive Age/ Serum Plasma Louisiana pine snakes (Pituophis sex ruthveni) in serum and plasma 17β-estradiol Progesterone Testosterone 17β-estradiol Progesterone Testosterone pmol/L nmol/L nmol/L pmol/L nmol/L nmol/L

AF 1364.47 1.02 <0.12 956.37 0.86 <0.12 AM 1030.12 2.04 35.53 711.81 1.02 40.81 AF 1116.39 < 0.64 < 0.12 946.86 < 0.64 < 0.12 AM 1058.28 < 0.64 8.33 810.08 <0.64 6.97 JF QNS QNS QNS 674.77 2.32 <0.12 JF 1073.91 2.19 <0.12 800.57 1.43 <0.12 JF QNS QNS QNS 675.57 0.70 <0.12 JF 948.66 0.76 <0.12 727.85 < 0.64 < 0.12 JM 859.93 1.08 <0.12 772.53 < 0.64 < 0.12

< 0.64 and < 0.12 are below detection limits for progesterone and testosterone, respectively. Data in italics were excluded from statistical analysis QNS, quantity not sufficient; A,adult;J, juvenile; F,female;M, male set of at least 20 but ideally 120 and appropriate statis- apparently healthy. In captive-bred cobras, PCV and lympho- tical methodology (Cray 2015; Geffre et al. 2009a; cyteswerehigherinwildcaughtcomparedwithcaptive-bred Geffre et al. 2009b). This can prove challenging given snakes, presumably due to nutritional and environmental fac- the often low availability of individuals in a given non- tors such as exposure to infectious agents and consequent domestic species, which is also a limitation of our immune stimulation (Salakij et al. 2002). Although our study study. Despite this limitation, we are using descriptive did not compare captive with wild-caught LPS, this conclu- methodology in order to provide baseline data that rep- sion may be applicable to our captive-bred group of LPS hav- resent one step in filling a knowledge gap on hematol- ing generally lower WBC. ogy, chemistry, and hormone data in this endangered Plasma biochemical differences in LPS were manifested as species. Very limited information on hematological data life stage but not sex differences and were similar to wild- are available for the other 4 species of the same genus caught, laboratory-housed gopher snakes (Dietz and Brodie Pituophis to date (Dietz and Brodie 1969; Mader et al. 1969; Mader et al. 1985). Variations of proteins and calcium 1985). are most consistent with physiological differences in juvenile Hematology data demonstrated life stage and sex differ- and adult LPS. The observed correlation of TS and TP indi- ences in LPS. The lower PCV in two juveniles with concurrent cates that initial evaluation of TS in in-house laboratories can evidence of erythroid regeneration indicate regenerative ane- be useful as an initial TP estimate, keeping the following con- mia of undetermined inciting cause as these two snakes were siderations in mind. As in other species, the refractive index otherwise clinically normal. Higher PCV in males compared may be affected by hemolysis, lipemia, and other chemical with females is common in reptile species and consistent with constituents, such as urea and electrolytes (Stockham 2008). physiological variation (Campbell 2015a; Wilkinson 2004). The biuret method for TP measurement is considered the gold PCV data of LPS was comparably higher than reported for standard for quantification of TP (Jacobson 1992). For accu- wild-caught, laboratory-housed gopher snakes (Dietz and rate quantification of protein fractions, including albumin and Brodie 1969; Mader et al. 1985). The significantly higher globulins, protein electrophoresis is required (Stockham 2008; lymphocytes in adult compared with juvenile snakes and in Zaias and Cray 2002). The observed higher calcium in adults adult male versus adult female snakes presumptively present a compared with juveniles presumably resulted from biological physiological difference. Such variations of leukocytes by life variation. Since these samples were collected outside the stage and sex are important to consider in hemogram interpre- breeding season, it was anticipated that females and males tation. Similar to other snake species, lymphocytes were the have similar concentrations of plasma analytes. predominant WBC type (Divers et al. 1996; Lamirande et al. There are limited studies on bile acid concentrations in 1999; Sypek and Borysenko 2009; Troiano et al. 1997). reptiles (Knotek et al. 2009; Knotkova et al. 2008; McBride Although azurophils reportedly are often the second most fre- et al. 2006), with fasting reptiles tending to have values lower quent WBC type in snakes (Alleman et al. 1992; Salakij et al. than 60 μmol/L (Divers 2000). Even though a significant dif- 2002; Troiano et al. 1997), our study animals had very low ference was noted between adults and juveniles in our study, numbers, which supports the clinical findings of snakes being all bile acid data were below 15 μmol/L. Further bile acid Comp Clin Pathol studies in healthy and diseased, fasted and non-fasted snakes, Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// and using appropriate assay validation for reptile species are creativecommons.org/licenses/by/4.0/), which permits unrestricted use, warranted. distribution, and reproduction in any medium, provided you give There are some limitations of the endocrine data of our appropriate credit to the original author(s) and the source, provide a link study, given the low number of study animals and data based to the Creative Commons license, and indicate if changes were made. on a single blood collection in all snakes and considering the complexity of reptile endocrinology and reproduction (Taylor and DeNardo 2011). 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Gen Comp Endocrinol 122:260–269 ences that reflect physiological variations and that are neces- Friedrichs KR, Harr KE, Freeman KP, Szladovits B, Walton RM, sary to consider in the interpretation of blood analyte data Barnhart KF, Blanco-Chavez J (2012) ASVCP reference interval from this species. The data presented herein will be useful guidelines: determination of de novo reference intervals in veteri- – for individual animals in managed care, provides an incentive nary species and other related topics. Vet Clin Pathol 41:441 453 Geffre A, Braun JP, Trumel C, Concordet D (2009a) Estimation of refer- for future investigations, and may have applications for future ence intervals from small samples: an example using canine plasma population-level assessments. creatinine. Vet Clin Pathol 38:477–484 Geffre A, Friedrichs K, Harr K, Concordet D, Trumel C, Braun JP Acknowledgments The authors thank Julie Fields, Jillene Sennon, and (2009b) Reference values: a review. 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